Single-cell nucleic acids for high-throughput studies

ABSTRACT

Described herein are cell-based analytic methods, including a method of incorporating nucleic acid sequences into reaction products from a cell population, wherein the nucleic acid sequences are incorporated into the reaction products of each cell individually or in small groups of cells individually. Also described herein is a matrix-type microfluidic device that permits at least two reagents to be delivered separately to each cell or group of cells, as well as primer combinations useful in the method and device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No.62/126,349, filed Feb. 27, 2015, which is hereby incorporated byreference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD

The subject matter disclosed herein relates to generally to the area ofanalysis of single cells or small groups of cells. In particular, thesubject matter relates to methods and compositions for performingseparate reactions with at least two reagents independently added tomultiple cells or groups of cells (or the components thereof),optionally followed by further analysis.

BACKGROUND

There is an interest in the life sciences in the quantitation oftranscription in substantial numbers of single cells in one study. Atpresent, Fluidigm Corporation enables the study of transcription from 96single cells at a time through the C1™ system “integrated fluidiccircuit” (IFC™) microfluidic devices.

Currently available chemistries from commercial sources used to preparecDNA from single cells for analysis of mRNA transcript levels, usuallybut not limited to mRNA sequencing, cannot be used in a Fluidigm IFC™unless there is an addressable outlet well for each single cell. Thereis no easy way to identify the transcripts from single cells using thecommercial kits when the cDNA from each cell is combined together in apool; this is necessary in Fluidigm IFCs™ developed for more than 96single cells on the Generation 2 carrier since there are not enoughwells to output each discrete single-cell cDNA sample.

If the cDNA is to be sequenced, e.g., using the bridge amplification(cluster generation) and sequencing method commercialized by Illumina,Inc. (San Diego, Calif.), a further problem is the need for individualcommercial tagmentation reactions for each cell's cDNA from the 96-cellIFC™ to allow for controlled fragmentation for flow cell clustering andsample identification during sequencing.

SUMMARY

This disclosure includes the development of a high throughput (HT)capture architecture in an IFC as well as companion chemistry, whichfacilitates convenient single cell transcriptome amplification andidentification of specific transcripts from each cell and has a varietyof other applications as well.

In various aspects, the disclosure(s) contemplated herein may include,but need not be limited to, any one or more of the followingembodiments:

Embodiment 1

A method of exposing cells from a population to at least two differentreagents, wherein each cell is exposed to the reagents individually, orin groups of two of more, the method including:

(a) distributing cells from the population to a plurality of capturesites in a microfluidic device so that a plurality of capture sites eachincludes one or more cells;

(b) providing one or more first reagent(s) to each capture site;

(c) providing one or more second reagent(s) to each capture site,wherein the second reagent(s) is/are different from the first reagent(s)and is/are provided separately from the first reagent(s);

(d) conducting a reaction, whereby the reaction products encode an itemof capture site information;

(e) recovering the reaction products; and

(f) analyzing the reaction products, wherein such analysis permits theidentification of particular reaction products as having been derivedfrom a single cell or group of cells at a particular capture site.

Embodiment 2

A method of incorporating nucleic acid sequences into reaction productsfrom a cell population, wherein the nucleic acid sequences areincorporated into the reaction products of each cell individually, or ingroups of up to 1000, the method including:

(a) distributing cells from the population to a plurality of capturesites in a microfluidic device so that a plurality of capture sites eachincludes one or more cells;

(b) providing one or more first reagent(s) to each capture site;

(c) providing one or more second reagent(s) to each capture site,wherein the second reagent(s) is/are different from the first reagent(s)and is/are provided separately from the first reagent(s);

(d) conducting a reaction in which nucleic acid sequences areincorporated into the reaction products of each cell or group of cells,individually;

(e) recovering the reaction products; and

(f) analyzing the reaction products, wherein such analysis permits theidentification of particular reaction products as having been derivedfrom a single cell or group of cells at a particular capture site.

Embodiment 3

A method of incorporating nucleic acid sequences into nucleic acids of acell population, wherein the nucleic acid sequences are incorporatedinto the nucleic acids of each cell individually or in groups of up to1000, the method including:

(a) distributing cells from the population to a plurality of capturesites in a microfluidic device so that a plurality of capture sites eachincludes one or more cells;

(b) providing one or more first reagent(s) to each capture site;

(c) providing one or more second reagent(s) to each capture site,wherein the second reagent(s) is/are different from the first reagent(s)and is/are provided separately from the first reagent(s);

(d) conducting a reaction in which nucleic acid sequences areincorporated into the nucleic acids of each cell or group of cells,individually, to produce reaction products;

(e) recovering the reaction products; and

(f) analyzing the reaction products, wherein such analysis permits theidentification of particular reaction products as having been derivedfrom a single cell or group of cells at a particular capture site.

Embodiment 4

The method of embodiment any preceding embodiments, where thedistribution is carried out so that a plurality of capture sites eachcomprise not more than a single cell.

Embodiment 5

The method of any preceding embodiment, wherein the reactionincorporates a nucleotide barcode into the reaction products.

Embodiment 6

The method of embodiment 5, wherein the barcode encodes an item ofcapture site information.

Embodiment 7

The method of any preceding embodiment, wherein the reactionincorporates a nucleic acid sequence that uniquely identifies themolecule into which it is incorporated (UMI).

Embodiment 8

The method of embodiment 3, wherein the reaction includes reversetranscription of RNA.

Embodiment 9

The method of embodiment 8, wherein the first reagent(s) comprise areverse transcription (RT) primer including a poly-dT sequence and afirst barcode 5′ of the poly-dT sequence.

Embodiment 10

The method of embodiment 9, wherein the RT primer additionally includesa first UMI.

Embodiment 11

The method of embodiment 10, wherein the first UMI is 5′ of the poly-dTsequence.

Embodiment 12

The method of embodiments 9-11, wherein the RT primer additionallyincludes a first linker.

Embodiment 13

The method of embodiment 12, wherein the first linker is at the 5′ endof the RT primer.

Embodiment 14

The method of embodiments 9-13, wherein the RT primer additionallyincludes an anchor sequence 3′ of the poly-dT sequence.

Embodiment 15

The method of embodiments 8-14, wherein the reaction additionallyincludes second-strand synthesis to produce cDNA.

Embodiment 16

The method of embodiments 8-15 wherein the second reagent(s) comprise a5′ oligonucleotide including a poly-riboG sequence.

Embodiment 17

The method of embodiment 15, wherein the 5′ oligonucleotide includes asecond barcode 5′ of the poly-riboG sequence.

Embodiment 18

The method of embodiments 15 or 17, wherein the 5′ oligonucleotideadditionally includes a second UMI.

Embodiment 19

The method of embodiment 18, wherein the second UMI is 5′ of thepoly-riboG sequence.

Embodiment 20

The method of 15-19, wherein the 5′ oligonucleotide additionallyincludes a second linker.

Embodiment 21

The method of embodiment 20, wherein the second linker is at the 5′ endof the 5′ oligonucleotide.

Embodiment 22

The method of embodiment 21, wherein the method includes producing cDNA,wherein one strand has the structure: 5′-second linker-nucleotidesequence derived from RNA-first linker-3′, with a barcode located inbetween the linkers.

Embodiment 23

The method of embodiment 22, wherein the first barcode is locatedadjacent to the first linker.

Embodiment 24

The method of embodiment 23, wherein the second barcode is locatedadjacent to the second linker.

Embodiment 25

The method of embodiment 23, wherein said one strand of cDNA has thestructure: 3′-second linker-poly dC-nucleotide sequence derived fromRNA-first barcode-first linker-5′.

Embodiment 26

The method of embodiment 25, wherein said one strand of cDNA has thestructure: 3′-second linker-second barcode-poly dC-nucleotide sequencederived from RNA-first barcode-first linker-5′.

Embodiment 27

The method of embodiment 25, wherein said one strand of cDNA has astructure selected from the group consisting of: 3′-second linker-polydC-nucleotide sequence derived from RNA-first UMI-first barcode-firstlinker-5′; and 3′-second linker-poly dC-nucleotide sequence derived fromRNA-first barcode-first UMI-first linker-5′.

Embodiment 28

The method of embodiments 27, wherein said one strand of cDNA has astructure selected from the group consisting of: 3′-second linker-secondbarcode-second UMI-poly dC-nucleotide sequence derived from RNA-firstUMI-first barcode-first linker-5′; 3′-second linker-secondbarcode-second UMI-poly dC-nucleotide sequence derived from RNA-firstbarcode-first UMI-first linker-5′; 3′-second linker-second UMI-secondbarcode-poly dC-nucleotide sequence derived from RNA-first UMI-firstbarcode-first linker-5′; and 3′-second linker-second UMI-secondbarcode-poly dC-nucleotide sequence derived from RNA-first barcode-firstUMI-first linker-5′.

Embodiment 29

The method of embodiment 3, wherein the reaction includes amplificationof DNA.

Embodiment 30

The method of embodiment 29, wherein the first and/or second reagent(s)comprise first and/or second amplification primers, respectively,wherein the first and/or second amplification primers comprise a firstor second barcode, respectively, that is 5′ of a primer sequence.

Embodiment 31

The method of embodiment 30, wherein the first and/or secondamplification primers additionally comprise a first or second UMI,respectively.

Embodiment 32

The method of embodiment 31, wherein the first or second UMI is 5′ ofthe primer sequence.

Embodiment 33

The method embodiments 30-32, wherein the first and/or secondamplification primer additionally includes a first or second linker.

Embodiment 34

The method of embodiment 33, wherein the first or second linker is atthe 5′ end of the amplification primer.

Embodiment 35

The method of embodiment 34, wherein the method includes producingamplicons, wherein one strand has the structure: 5′-secondlinker-nucleotide sequence derived from cellular DNA-first linker-3′,with a barcode located in between the linkers.

Embodiment 36

The method of embodiment 35, wherein one strand has the structure:3′-second linker-nucleotide sequence derived from cellular DNA-firstbarcode-first linker-5′.

Embodiment 37

The method of embodiment 25, wherein said one strand has the structure:3′-second linker-second barcode-nucleotide sequence derived fromcellular DNA-first barcode-first linker-5′.

Embodiment 38

The method of embodiment 25, wherein said one strand has a structureselected from the group consisting of: 3′-second linker-nucleotidesequence derived from cellular DNA-first UMI-first barcode-firstlinker-5′; and 3′-second linker-nucleotide sequence derived fromcellular DNA-first barcode-first UMI-first linker-5′.

Embodiment 39

The method of embodiments 27, wherein said one strand has a structureselected from the group consisting of: 3′-second linker-secondbarcode-second UMI-nucleotide sequence derived from cellular DNA-firstUMI-first barcode-first linker-5′; 3′-second linker-secondbarcode-second UMI-nucleotide sequence derived from cellular DNA-firstbarcode-first UMI-first linker-5′; 3′-second linker-second UMI-secondbarcode-nucleotide sequence derived from cellular DNA-first UMI-firstbarcode-first linker-5′; and 3′-second linker-second UMI-secondbarcode-poly dC-nucleotide sequence derived from cellular DNA-firstbarcode-first UMI-first linker-5′.

Embodiment 40

The method of any preceding embodiment, wherein the microfluidic deviceincludes a matrix-type microfluidic device including: capture sitesarranged in a matrix of R rows and C columns, wherein R and C areintegers greater than 1, and wherein the capture sites can befluidically isolated from one another after distribution of cells to thecapture sites; a set of R first input lines configured to deliver thefirst reagent(s) to capture sites in a particular row; a set of C secondinput lines configured to deliver second reagent(s) to capture sites ina particular column, wherein said delivery is separate from the deliveryfirst reagent(s), wherein, after the reaction, reaction products arerecovered from the microfluidic device in pools of reaction productsfrom individual rows or columns.

Embodiment 41

The method of embodiment 40, wherein an RT primer is delivered tocapture sites via one set of the input lines, and a 5′ oligonucleotideis delivered to the capture sites via the other set of input lines.

Embodiment 42

The method of embodiment 40, wherein a first amplification primer isdelivered to capture sites via one set of the input lines, and a secondamplification primer is delivered to the capture sites via the other setof input lines.

Embodiment 43

The method of any preceding embodiment, wherein all methods steps areperformed in the microfluidic device.

Embodiment 44

The method of any of the preceding embodiments, wherein the reactionproducts are subjected to preamplification using linker primers thatanneal to the first and second linkers, wherein the linker primers arethe same or different.

Embodiment 45

The method of embodiment 44, wherein said preamplification is performedin the microfluidic device.

Embodiment 46

The method of any of the preceding embodiments, wherein the reactionproducts are subjected to tagmentation.

Embodiment 47

The method of any preceding embodiment, wherein the reactionincorporates one or more DNA sequencing primer binding sites into thereaction products.

Embodiment 48

The method of any of the preceding embodiments, wherein the reactionproducts are subjected to DNA sequencing.

Embodiment 49

The method of embodiment 48, wherein the sequences obtained from DNAsequencing are identified as having been derived from a particularcapture site based on one or two barcodes.

Embodiment 50

The method of any of embodiments 40-49, wherein the exported pools areseparately subjected to one or more of the steps of embodiments 44-49.

Embodiment 51

The method of any of embodiments 40-49, wherein the exported pools arecombined into one reaction mixture, which is subjected to one or more ofthe steps of embodiments 44-49.

Embodiment 52

The method of any of embodiments 40-51, wherein the microfluidic deviceis sufficiently transparent on at least one surface to permitvisualization of cells and/or, when a visualizable label is employed,signals associated with cells or reaction products.

Embodiment 53

The method of embodiment 52, additionally including imaging thecell-occupied capture sites before conducting the reaction.

Embodiment 54

The method of any preceding embodiment, wherein the reaction includeswhole transcriptome amplification (WTA), whole genome amplification(WGA), protein proximity ligation, microRNA (mRNA) preamplification,target-specific amplification of RNA or DNA.

Embodiment 55

The method of any preceding embodiment, wherein the microfluidic deviceincludes at least 750 capture sites.

Embodiment 56

A matrix-type microfluidic device including:

a plurality of capture sites arranged in a matrix of R rows and Ccolumns, wherein R and C are integers greater than 1, and wherein:

-   -   each capture site includes a capture feature that captures one        or more cells;    -   the capture sites can be fluidically isolated from one another        after distribution of cells to the capture sites;

a set of R first input lines configured to deliver the first reagent(s)to capture sites in a particular row; and

a set of C second input lines configured to deliver second reagent(s) tocapture sites in a particular column, wherein said delivery is separatefrom the delivery first reagent(s).

Embodiment 57

The device of embodiment 56, wherein the capture feature is configuredto capture not more than a single cell.

Embodiment 58

The device of embodiments 56 or 57, wherein the microfluidic device issufficiently transparent on at least one surface to permit visualizationof cells and/or, when a visualizable label is employed, signalsassociated with cells or reaction products.

Embodiment 59

The device embodiments 56-58, wherein each capture site includes fourchambers that can be fluidically isolated from one another, wherein oneof said chambers includes the capture feature.

Embodiment 60

A method of operating the microfluidic device of embodiments 56-59,wherein the method includes:

(a) distributing cells from a population of cells to the capture sitesso that a plurality of capture sites comprise one or more cells;

(b) after distribution, fluidically isolating the capture sites from oneanother;

(c) providing one or more first reagent(s) to each fluidically isolatedcapture site via the R first input lines;

(d) providing one or more second reagent(s) to each fluidically isolatedcapture site via the C second input lines, wherein the second reagent(s)is/are different from the first reagent(s); and

(e) conducting a reaction.

Embodiment 61

The method of embodiment 60, wherein a plurality of capture sitescomprise not more than a single cell.

Embodiment 62

The method of embodiments 60 or 61, additionally including recoveringthe reaction products as a pool of reaction products from each row or asa pool of reaction products from each column.

Embodiment 63

The method of embodiments 60-62, wherein said recovering includesproviding a harvesting reagent to the R first input lines or the Csecond input lines.

Embodiment 64

A primer combination for use in producing cDNA from RNA, the combinationincluding:

(a) a reverse transcription (RT) primer including an anchor sequence, apoly-dT sequence 5′ of the anchor sequence, a first barcode 5′ of thepoly-dT sequence, and a first linker 5′ of the first barcode sequence;and

(b) a 5′ oligonucleotide including a poly-riboG sequence, a secondbarcode 5′ of the poly-riboG sequence, and a second linker 5′ of thesecond barcode.

Embodiment 65

The primer combination of embodiment 64, wherein one or both primerscomprise a UMI.

Embodiment 66

A primer combination for use in amplifying DNA, the combinationincluding first and second amplification primers that can prime theproduction of an amplicon in the presence of suitable template DNA,wherein each amplification primer includes: a primer sequence; a barcodethat is 5′ of the primer sequence, wherein the barcodes in each primerare different; and a linker that is 5′ of the barcode; wherein one orboth primers also comprise a UMI that is 5′ of the primer sequence and3′ of the linker.

Embodiment 67

The primer combination of embodiments 64-66, additionally including oneor more linker primer(s) that anneal(s) to the linkers.

Embodiment 68

The primer combination of embodiments 64-67, wherein the linker primerscomprise a 5′ linker primer and a different 3′ linker primer.

Embodiment 69

The primer combination of embodiments 68, additionally including aprimer including a portion specific for the 3′ linker primer or itscomplement and/or a primer including a portion specific for the 5′linker primer or its complement, wherein the primer(s) additionallycomprise a flow cell sequence useful in cluster generation in bridgesequencing.

Embodiment 70

A method of producing cDNA from RNA, wherein the primer combination ofembodiments 64 or 65 is employed for first-strand synthesis.

Embodiment 71

A method of amplifying DNA, the method including contacting template DNAwith the primer combination of embodiment 66 to produce amplicons.

Embodiment 72

A method of preamplifying the cDNA or amplicons of embodiments 70 or 71,respectively, the method including preamplifying the cDNA or ampliconswith the linker primers of embodiments 67 and 68.

Embodiment 73

A method of cluster generation in bridge sequencing of cDNA or ampliconsproduced in embodiments 70-72, the method conducting cluster generationusing the primer of embodiment 69.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-D: An illustrative matrix-type microfluidic device is shownschematically in (A). (B) illustrates the delivery of R differentbarcodes through the R different first input lines to the capture sites.(C) illustrates the delivery of C different barcodes through C differentinput lines to the capture sites. (D) illustrates that, after thereaction has been carried out, reaction products can be harvested foreach column as a pool, for example, by applying a harvesting fluid tothe C second input lines to push the reaction products out of outlets atone end of the input lines.

FIG. 2: A photograph of the illustrative matrix-type microfluidic deviceshown schematically in FIG. 1.

FIG. 3: A photomicrograph of an illustrative capture feature suitablefor capturing a single cell in a matrix-type microfluidic device, aswell as an illustrative flowchart for a method in which barcodes areadded by row and column into the reaction products from the capturedcell.

FIG. 4A-E: (A) shows a series of increasingly more miniaturizedillustrative capture features, which facilitate the analysis of morecells per microfluidic device. (B) shows the arrangement of capturefeatures in rows and columns, together with the channels fordistributing cells to the capture features. (C) shows photomicrographsof the miniaturized capture features. (D) reports the cell occupancydata for two different capture site designs, with the top panel of thetable showing the results for the miniaturized features, and the bottompanel of the table showing the results for illustrative double groovescapture features with different height ratios for top and bottomgrooves. The first column in the table reports the number of capturefeatures having no cells, and the second column reports the number ofcapture features having just 1 cell. (E) shows a schematic of an IFChaving two different capture site designs: one having reduced dimensionsand one having different height ratios for top and bottom grooves.

FIG. 5: A schematic of illustrative double grooves capture featuresincluding the channels for distributing cells to the capture features.This geometry allows for more capture sites per unit area.

FIG. 6A-D: (A) shows the flow resistance for the main and bypass flow inan illustrative double grooves capture device, illustrated schematicallyin (B) and in photomicrographs in (C) and (D).

FIG. 7A-B shows the activation of bypass peristaltic pumping in anillustrative double grooves capture device, schematically in (A) and ina photomicrograph in (B).

FIG. 8A-C: (A) and (B) illustrate closing the main flow output in anillustrative double grooves capture device, schematically in (A) and ina photomicrograph (B). (C) reports cell occupancy data for the doublegrooves capture feature design, with number of capture features havingno cells in column 1 of the table, and the number of capture featureshaving just 1 cell in column 2 of the table.

FIG. 9: An illustrative unit capture site with four chambers, each ofwhich is available for reagent(s). The capture feature is located in oneof these four chambers. In use, a first reagent(s) can be loaded intothe capture site from bottom to top (purple arrow), and a second reagentcan be loaded into a capture site from left to right (green arrow).

FIG. 10: A scheme for single-cell transcriptome identification (formessenger [poly-A] RNA) and 3′ end counting of the transcripts, which isdescribed in Example 2.

FIG. 11: An illustrative capture site suitable for practicing thesingle-cell transcriptome identification scheme of FIG. 10. Workflow isshown above a photomicrograph labeled to show four chambers, separatedby 4 valves (a-e), as well as fluid flow through the capture site (1-7).

FIG. 12 shows the same scheme for single-cell transcriptomeidentification as FIG. 10 to the right of a proposed layout for fourchambers of a capture site.

FIG. 13: An illustrative scheme for combinatorial barcoding of nucleicacid (BC1+2=two different barcodes; Univ1 and Univ2=universal primerbinding sites, which can be the same or different).

FIG. 14: An illustrative scheme for target-specific amplification ofRNA, with combinatorial barcoding, followed by paired-end sequencing.

FIG. 15: An illustrative scheme for combinatorial barcoding of nucleicacid by ligation (BC1+2=two different barcodes; Univ1 andUniv2=universal primer binding sites, which can be the same ordifferent).

FIG. 16: An illustrative scheme for using combinatorial barcoding inprotein proximity ligation (PLA) or extension (PEA; BC1+2=two differentbarcodes; Univ1 and Univ2=universal primer binding sites, which can bethe same or different).

FIG. 17: An illustrative scheme for combinatorial barcoding using anenzyme, such as a transposase, to introduce the barcodes (BC1+2=twodifferent barcodes; Univ1 and Univ2=universal primer binding sites,which can be the same or different).

FIG. 18: Illustrative data from a sequencing study in which sequenceswere barcoded on a matrix-type microfluidic device, followed bysequencing, and “demultiplexing” using column and row barcode toattribute particular sequencing reads to particular capture sites.

FIG. 19: Illustrative data from a sequencing study in which sequenceswere barcoded on a matrix-type microfluidic device, followed bysequencing, and “demultiplexing” using column and row barcode toattribute particular sequencing reads to particular capture sites.

DETAILED DESCRIPTION

Described herein is a hygienic barcoding strategy on a HT IFC™ to allowfor pooling of cDNA from many single cells (or small numbers of cells)post-IFC™ where the cells can be de-multiplexed from one another usingcell-specific barcodes on each molecule following analysis of thetranscripts. This strategy can be designed to facilitate simple barcodeenrichment by amplification so that the majority of material queriedwill be barcoded rather than cDNA material that cannot be attributed toa particular cell, leading to generation of unusable sequence data.

Other barcoded cDNA enrichment strategies for single cells involvecustom-made transposons and biotin-strepavidin-pulldowns increasing theworkflow complexity of the enrichment while at the same time potentiallyreducing the amount of material available for sequencing due to thenature of this type of cleanup. The amplification-based barcodeenrichment strategy enables single tagmentation reactions for largenumbers of cells instead of one cell at a time without pulldowns orextra cleanup steps and without the need for generation of customtransposons.

Also described herein is an IFC™ architecture that enables theprocessing of discreetly captured or isolated single cells (or groups ofcells) in combination with any multistep biochemical process thatfacilitates the analysis of intracellular macromolecules. Such processinclude, but are not limited to, Whole Genome amplification (WGA) forDNA sequencing, multiplexed protein proximity ligation assays toquantitate specific proteins, multiplexed microRNA preamplication,target-specific amplification of RNA transcripts or DNA sequences (e.g.,genotyping polymorphic markers, such as SNPs, or otherwise analyzinggenetic variations, such as copy number variations), targetedresequencing, or any combination thereof. In addition using the novelIFC™ architecture, simple modifications to the IFC™-associated controlscripts enable the real-time detection of transcripts of single cells,which can be used in combination, for example, a controller that enablesintegrated thermal and pneumatic control, with optical detection of allunit cells in tandem. This combination provides the ability to linkphenotype with gene expression analysis, while eliminating the lengthyand undesirable off-instrument imaging steps. This architecture can alsobe exploited to culture discreetly captured/isolated cells or groups ofcells under any desired conditions, which can be modified on-chip byadding components such as, e.g., agonists or antagonists for particularreceptors.

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified. These terms are defined specificallyfor clarity, but all of the definitions are consistent with how askilled artisan would understand these terms.

As used herein, the term “microfluidic device” refers to any device thatincludes chambers and/or channels wherein at least one dimension is lessthan 1 millimeter. In certain embodiments, a microfluidic deviceincludes fluid flow channels (or lines) and separate control channels(or lines) that function to control or regulate flow through the fluidchannels.

The term nucleic acid includes any form of DNA or RNA, including, forexample, genomic DNA; complementary DNA (cDNA), which is a DNArepresentation of mRNA, usually obtained by reverse transcription ofmessenger RNA (mRNA) or by amplification; DNA molecules producedsynthetically or by amplification; and mRNA.

The term nucleic acid encompasses double- or triple-stranded nucleicacids, as well as single-stranded molecules. In double- ortriple-stranded nucleic acids, the nucleic acid strands need not becoextensive (i.e., a double-stranded nucleic acid need not bedouble-stranded along the entire length of both strands).

The term nucleic acid also encompasses any chemical modificationthereof, such as by methylation and/or by capping. Nucleic acidmodifications can include addition of chemical groups that incorporateadditional charge, polarizability, hydrogen bonding, electrostaticinteraction, and functionality to the individual nucleic acid bases orto the nucleic acid as a whole. Such modifications may include basemodifications such as 2′-position sugar modifications, 5-positionpyrimidine modifications, 8-position purine modifications, modificationsat cytosine exocyclic amines, substitutions of 5-bromo-uracil, backbonemodifications, unusual base pairing combinations such as the isobasesisocytidine and isoguanidine, and the like.

More particularly, in certain embodiments, nucleic acids, can includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and any other type of nucleicacid that is an N- or C-glycoside of a purine or pyrimidine base, aswell as other polymers containing nonnucleotidic backbones, for example,polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino(commercially available from the Anti-Virals, Inc., Corvallis, Oreg., asNeugene) polymers, and other synthetic sequence-specific nucleic acidpolymers providing that the polymers contain nucleobases in aconfiguration which allows for base pairing and base stacking, such asis found in DNA and RNA. The term nucleic acid also encompasses linkednucleic acids (LNAs), which are described in U.S. Pat. Nos. 6,794,499,6,670,461, 6,262,490, and 6,770,748, which are incorporated herein byreference in their entirety for their disclosure of LNAs.

The nucleic acid(s) can be derived from a completely chemical synthesisprocess, such as a solid phase-mediated chemical synthesis, from abiological source, such as through isolation from any species thatproduces nucleic acid, or from processes that involve the manipulationof nucleic acids by molecular biology tools, such as DNA replication,PCR amplification, reverse transcription, or from a combination of thoseprocesses.

The term “template” is used herein to refer to a nucleic acid moleculethat serves as a template for a polymerase to synthesize a complementarynucleic acid molecule.

There term “template nucleic acids” is a generic term that encompasses“target nucleic acids.”

The term “target nucleic acids” is used herein to refer to particularnucleic acids to be detected in the methods described herein.Accordingly, amplification of single nucleotide polymorphisms (SNPs),for example, is an example of target-specific amplification, whereaswhole genome amplification is an example of the amplification that aimsto amplify all template nucleic acids in the genome.

As used herein, the term “target nucleotide sequence” refers to amolecule that includes the nucleotide sequence of a target nucleic acid,such as, for example, the amplification product obtained by amplifying atarget nucleic acid or the cDNA produced upon reverse transcription ofan RNA target nucleic acid.

As used herein, the term “complementary” refers to the capacity forprecise pairing between two nucleotides. I.e., if a nucleotide at agiven position of a nucleic acid is capable of hydrogen bonding with anucleotide of another nucleic acid, then the two nucleic acids areconsidered to be complementary to one another at that position.Complementarity between two single-stranded nucleic acid molecules maybe “partial,” in which only some of the nucleotides bind, or it may becomplete when total complementarity exists between the single-strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. A first nucleotide sequence is said to bethe “complement” of a second sequence if the first nucleotide sequenceis complementary to the second nucleotide sequence. A first nucleotidesequence is said to be the “reverse complement” of a second sequence, ifthe first nucleotide sequence is complementary to a sequence that is thereverse (i.e., the order of the nucleotides is reversed) of the secondsequence.

“Specific hybridization” refers to the binding of a nucleic acid to atarget nucleotide sequence in the absence of substantial binding toother nucleotide sequences present in the hybridization mixture underdefined stringency conditions. Those of skill in the art recognize thatrelaxing the stringency of the hybridization conditions allows sequencemismatches to be tolerated.

In particular embodiments, hybridizations are carried out understringent hybridization conditions. The phrase “stringent hybridizationconditions” generally refers to a temperature in a range from about 5°C. to about 20° C. or 25° C. below than the melting temperature (T_(m))for a specific sequence at a defined ionic strength and pH. As usedherein, the T_(m) is the temperature at which a population ofdouble-stranded nucleic acid molecules becomes half-dissociated intosingle strands. Methods for calculating the T_(m) of nucleic acids arewell known in the art (see, e.g., Berger and Kimmel (1987) METHODS INENZYMOLOGY, VOL. 152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego:Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: ALABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory),both incorporated herein by reference). As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative FilterHybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The meltingtemperature of a hybrid (and thus the conditions for stringenthybridization) is affected by various factors such as the length andnature (DNA, RNA, base composition) of the primer or probe and nature ofthe target nucleic acid (DNA, RNA, base composition, present in solutionor immobilized, and the like), as well as the concentration of salts andother components (e.g., the presence or absence of formamide, dextransulfate, polyethylene glycol). The effects of these factors are wellknown and are discussed in standard references in the art. Illustrativestringent conditions suitable for achieving specific hybridization ofmost sequences are: a temperature of at least about 60° C. and a saltconcentration of about 0.2 molar at pH7.

The term “oligonucleotide” is used to refer to a nucleic acid that isrelatively short, generally shorter than 200 nucleotides, moreparticularly, shorter than 100 nucleotides, most particularly, shorterthan 50 nucleotides. Typically, oligonucleotides are single-stranded DNAmolecules.

The term “primer” refers to an oligonucleotide that is capable ofhybridizing (also termed “annealing”) with a nucleic acid and serving asan initiation site for nucleotide (RNA or DNA) polymerization underappropriate conditions (i.e., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as DNA orRNA polymerase or reverse transcriptase) in an appropriate buffer and ata suitable temperature. The term “primer site” or “primer binding site”refers to the segment of the target nucleic acid to which a primerhybridizes.

A primer is said to anneal to another nucleic acid if the primer, or aportion thereof, hybridizes to a nucleotide sequence within the nucleicacid. The statement that a primer hybridizes to a particular nucleotidesequence is not intended to imply that the primer hybridizes eithercompletely or exclusively to that nucleotide sequence.

The term “primer pair” refers to a set of primers including a 5′“upstream primer” or “forward primer” that hybridizes with thecomplement of the 5′ end of the DNA sequence to be amplified and a 3′“downstream primer” or “reverse primer” that hybridizes with the 3′ endof the sequence to be amplified. As will be recognized by those of skillin the art, the terms “upstream” and “downstream” or “forward” and“reverse” are not intended to be limiting, but rather provideillustrative orientation in particular embodiments.

The primer or probe can be perfectly complementary to the target nucleicacid sequence or can be less than perfectly complementary. In certainembodiments, the primer has at least 65% identity to the complement ofthe target nucleic acid sequence over a sequence of at least 7nucleotides, more typically over a sequence in the range of 10-30nucleotides, and often over a sequence of at least 14-25 nucleotides,and more often has at least 75% identity, at least 85% identity, atleast 90% identity, or at least 95%, 96%, 97%. 98%, or 99% identity. Itwill be understood that certain bases (e.g., the 3′ base of a primer)are generally desirably perfectly complementary to corresponding basesof the target nucleic acid sequence. Primer and probes typically annealto the target sequence under stringent hybridization conditions.

As used herein the terms “nucleotide barcode” and “barcode” refer to aspecific nucleotide sequence that encodes information about cDNAproduced when a barcoded primer or oligonucleotide is employed inreverse transcription or the amplicon produced when one or more barcodedprimer(s) is/are employed in an amplification reaction. As shown inFIGS. 16 and 17, barcodes may also be added to nucleotide sequences byother means, such as by ligation or via transposases.

In some embodiments, the barcode encodes “an item of capture siteinformation.” For example, for reactions carried out on a matrix-typemicrofluidic device, a barcode can encode the row or column of a capturesite. Two barcodes, one encoding the row in which the barcode isintroduced and the other encoding the column in which that barcode isintroduced can define the specific capture site residing at theintersection of the row and column identified by the barcodes.

As used herein, “UMI” is an acronym for “unique molecular index,” alsoreferred to as “molecular index.” A UMI is one in a group of indexes inwhich each index (or barcode) has an index sequence that is differentfrom any of the other indexes in the group. One way to achieve this“uniqueness” is to use a string of nucleotides. For example, if thelength of this string is 10 bases, there are more than 1 million uniquesequences; if it is 20 bases long, there will be 10¹² unique sequences.See Hug and Schulernz, “Measurement of the Number of Molecules of aSingle mRNA Species in a Complex mRNA Preparation,” J. Theor. Biol.(2003) 221, 615-624 and Hollas and Schuler, “A Stochastic Approach toCount RNA Molecules Using DNA Sequencing Methods” in Algorithms inBioinformatics (2003): Third International Workshop, WABI 2003,Budapest, Hungary, Sep. 15-20, 2003, Series title: Lecture Notes inComputer Science Volume 2812, pp 55-62 (eds. Benson and Page).

A “linker” can, but need not, be or include a nucleic acid. Nucleotidelinkers can be added to either end of a nucleotide sequence to beamplified to facilitate unbiased amplification using primers specificfor the nucleotide linkers, which can be the same or different.

As used herein, an “anchor sequence” refers to a sequence in anoligonucleotide that serves to lock onto a target sequence, typicallyfollowing a stretch of identical nucleotide bases. It usually occurs onthe 3′ end of the oligonucleotide, but is not limited to this position.It can consist of random nucleotides, often excluding the nucleotidesfrom the stretch. For example, an illustrative anchor sequence thatfollows a poly-dT stretch in a primer (or oligonucleotide) might consistof a first position or portion containing any or all of the bases A, G,and/or C, but not T. The second position or portion might contain anycombination of bases or all of the bases (A, G, T, C) on the terminus ofthe primer or oligonucleotide.

The term “adjacent,” when used herein to refer two nucleotide sequencesin a nucleic acid, can refer to nucleotide sequences that are separatedby 1 to about 50 nucleotides, more specifically, by a range of about 1to about 20 nucleotides, even more specifically, by a range of about 1to about 10 nucleotides, or to sequences that directly abut one another(separated by 0 nucleotides).

As used herein with reference to a portion of a primer, the term“target-specific” nucleotide sequence refers to a sequence that canspecifically anneal to a target nucleic acid or a target nucleotidesequence under suitable annealing conditions. Portions of primers can be“specific” in the same sense for nucleotide sequences other thantargets.

Amplification according to the present teachings encompasses any meansby which at least a part of at least one target nucleic acid isreproduced, typically in a template-dependent manner, including withoutlimitation, a broad range of techniques for amplifying nucleic acidsequences, either linearly or exponentially. Illustrative means forperforming an amplifying step include ligase chain reaction (LCR),ligase detection reaction (LDR), ligation followed by Q-replicaseamplification, PCR, primer extension, strand displacement amplification(SDA), hyperbranched strand displacement amplification, multipledisplacement amplification (MDA), nucleic acid strand-basedamplification (NASBA), two-step multiplexed amplifications, rollingcircle amplification (RCA), and the like, including multiplex versionsand combinations thereof, for example but not limited to, OLA/PCR,PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known ascombined chain reaction—CCR), and the like. Descriptions of suchtechniques can be found in, among other sources, Ausbel et al.; PCRPrimer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press(1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih etal., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid ProtocolsHandbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson etal., Curr Opin Biotechnol. 1993 February; 4(1):41-7, U.S. Pat. No.6,027,998; U.S. Pat. No. 6,605,451, Barany et al., PCT Publication No.WO 97/31256; Wenz et al., PCT Publication No. WO 01/92579; Day et al.,Genomics, 29(1): 152-162 (1995), Ehrlich et al., Science 252:1643-50(1991); Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press (1990); Favis et al., Nature Biotechnology18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000);Belgrader, Barany, and Lubin, Development of a Multiplex LigationDetection Reaction DNA Typing Assay, Sixth International Symposium onHuman Identification, 1995 (available on the world wide web at:promega.com/geneticidproc/ussymp6proc/blegrad.html-); LCR KitInstruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002;Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook,Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res.27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66(2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl.Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002);Lage et al., Genome Res. 2003 February; 13(2):294-307, and Landegren etal., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002November; 2(6):542-8., Cook et al., J Microbiol Methods. 2003 May;53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February;12(1):21-7, U.S. Pat. No. 5,830,711, U.S. Pat. No. 6,027,889, U.S. Pat.No. 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No.WO9803673A1.

In some embodiments, amplification comprises at least one cycle of thesequential procedures of: annealing at least one primer withcomplementary or substantially complementary sequences in at least onetarget nucleic acid; synthesizing at least one strand of nucleotides ina template-dependent manner using a polymerase; and denaturing thenewly-formed nucleic acid duplex to separate the strands. The cycle mayor may not be repeated. Amplification can comprise thermocycling or canbe performed isothermally.

“Whole transcriptome amplification” (“WTA”) refers to any amplificationmethod that aims to produce an amplification product that isrepresentative of a population of RNA from the cell from which it wasprepared. An illustrative WTA method entails production of cDNA bearinglinkers on either end that facilitate unbiased amplification. In manyimplementations, WTA is carried out to analyze messenger (poly-A) RNA(this is also referred to as “RNAseq”).

“Whole genome amplification” (“WGA”) refers to any amplification methodthat aims to produce an amplification product that is representative ofthe genome from which it was amplified. Illustrative WGA methods includePrimer extension PCR (PEP) and improved PEP (I-PEP), Degeneratedoligonucleotide primed PCR (DOP-PCR), Ligation-mediated PCR (LMP),T7-based linear amplification of DNA (TLAD), Multiple displacementamplification (MDA).

The term “substantially” as used herein with reference to a parametermeans that the parameter is sufficient to provide a useful result. Thus,“substantially complementary,” as applied to nucleic acid sequencesgenerally means sufficiently complementary to work in the describedcontext. Typically, substantially complementary means sufficientlycomplementary to hybridize under the conditions employed.

A “reagent” refers broadly to any agent used in a reaction, other thanthe analyte (e.g., nucleic acid being analyzed). Illustrative reagentsfor a nucleic acid amplification reaction include, but are not limitedto, buffer, metal ions, polymerase, reverse transcriptase, primers,nucleotides, oligonucleotides, labels, dyes, nucleases, and the like.Reagents for enzyme reactions include, for example, substrates,cofactors, buffer, metal ions, inhibitors, and activators. The termreagent also encompasses any component that influences cell growth orbehavior, such as, e.g., buffer, culture medium or components thereof,agonists or antagonists, etc.

The term “label,” as used herein, refers to any atom or molecule thatcan be used to provide a detectable and/or quantifiable signal. Inparticular, the label can be attached, directly or indirectly, to anucleic acid or protein. Suitable labels that can be attached to probesinclude, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,spin labels, molecules that emit chemiluminescence, electrochemicallyactive molecules, enzymes, cofactors, and enzyme substrates.

The term “stain”, as used herein, generally refers to any organic orinorganic molecule that binds to a component to facilitate detection ofthat component.

The term “dye,” as used herein, generally refers to any organic orinorganic molecule that absorbs electromagnetic radiation at awavelength greater than or equal 340 nm.

The term “fluorescent dye,” as used herein, generally refers to any dyethat emits electromagnetic radiation of longer wavelength by afluorescent mechanism upon irradiation by a source of electromagneticradiation, such as a lamp, a photodiode, or a laser.

As use herein, the term “variation” is used to refer to any difference.A variation can refer to a difference between individuals orpopulations. A variation encompasses a difference from a common ornormal situation. Thus, a “copy number variation” or “mutation” canrefer to a difference from a common or normal copy number or nucleotidesequence. An “expression level variation” or “splice variant” can referto an expression level or RNA or protein that differs from the common ornormal expression level or RNA or protein for a particular, cell ortissue, developmental stage, condition, etc.

A “polymorphic marker” or “polymorphic site” is a locus at whichnucleotide sequence divergence occurs. Illustrative markers have atleast two alleles, each occurring at frequency of greater than 1%, andmore typically greater than 10% or 20% of a selected population. Apolymorphic site may be as small as one base pair. Polymorphic markersinclude restriction fragment length polymorphism (RFLPs), variablenumber of tandem repeats (VNTR's), hypervariable regions,minisatellites, dinucleotide repeats, trinucleotide repeats,tetranucleotide repeats, simple sequence repeats, deletions, andinsertion elements such as Alu. The first identified allelic form isarbitrarily designated as the reference form and other allelic forms aredesignated as alternative or variant alleles. The allelic form occurringmost frequently in a selected population is sometimes referred to as thewildtype form. Diploid organisms may be homozygous or heterozygous forallelic forms. A diallelic polymorphism has two forms. A triallelicpolymorphism has three forms.

A “single nucleotide polymorphism” (SNP) occurs at a polymorphic siteoccupied by a single nucleotide, which is the site of variation betweenallelic sequences. The site is usually preceded by and followed byhighly conserved sequences of the allele (e.g., sequences that vary inless than 1/100 or 1/1000 members of the populations). A SNP usuallyarises due to substitution of one nucleotide for another at thepolymorphic site. A transition is the replacement of one purine byanother purine or one pyrimidine by another pyrimidine. A transversionis the replacement of a purine by a pyrimidine or vice versa. SNPs canalso arise from a deletion of a nucleotide or an insertion of anucleotide relative to a reference allele.

As used herein with respect to reactions, reaction mixtures, reactionvolumes, etc., the term “separate” refers to reactions, reactionmixtures, reaction volumes, etc., where reactions are carried out inisolation from other reactions. Separate reactions, reaction mixtures,reaction volumes, etc. include those carried out in droplets (See, e.g.,U.S. Pat. No. 7,294,503, issued Nov. 13, 2007 to Quake et al., entitled“Microfabricated crossflow devices and methods,” which is incorporatedherein by reference in its entirety and specifically for its descriptionof devices and methods for forming and analyzing droplets; U.S. PatentPublication No. 20100022414, published Jan. 28, 2010, by Link et al.,entitled “Droplet libraries,” which is incorporated herein by referencein its entirety and specifically for its description of devices andmethods for forming and analyzing droplets; and U.S. Patent PublicationNo. 20110000560, published Jan. 6, 2011, by Miller et al., entitled“Manipulation of Microfluidic Droplets,” which is incorporated herein byreference in its entirety and specifically for its description ofdevices and methods for forming and analyzing droplets.), which may, butneed not, be in an emulsion, as well as those wherein reactions,reaction mixtures, reaction volumes, etc. are separated by mechanicalbarriers, e.g., separate vessels, separate wells of a microtiter plate,or separate compartments of a matrix-type microfluidic device.

The term “fluidically isolated” is used herein to refer to state inwhich two or more elements of a microfluidic device are not in fluidcommunication with one another.

The term “elastomer” has the general meaning used in the art. Thus, forexample, Allcock et al. (Contemporary Polymer Chemistry, 2nd Ed.)describes elastomers in general as polymers existing at a temperaturebetween their glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed.

Cell-Based Analytic Methods

Described herein is a method of exposing cells from a population to atleast two different reagents, wherein each cell is exposed to thereagents individually, or in groups of two of more. The method entailsdistributing cells from the population to a plurality of capture sitesin a microfluidic device so that a plurality of capture sites each hasone or more captured or isolated cells. In various embodiments, thecapture sites have groups of 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200,300, 400, 500, 600, 700, 800, 900, or 1000 cell(s) or groups having anumber of cells within a range defined by any of these values. In someembodiments, these values are defined by taking an average of the numberof cells per capture site.

One or more first reagent(s) is provided to each capture site, and oneor more second reagent(s) is provided to each capture site, wherein thesecond reagent(s) is/are different from the first reagent(s) and is/areprovided separately from the first reagent(s). Each pair of reagentscan, for example, be provided to a pair of fluidically isolatablechambers in the capture site that are distinct from one another and,optionally, distinct from a chamber including the capture feature.

In some embodiments, at least one surface of the microfluidic device istransparent to permit visualization of the cell or a signal from alabel. In such embodiments, the method can optionally include imagingthe cell-occupied capture sites before conducting the reaction.

One or more of the reagents can be an agent that supports cellulargrowth, modulates cellular behavior, and/or facilitates detection of acellular component (whether on the surface of the cell orintracellular). Indeed, the reagent can be any molecule or compositionthat one might wish to contact with a cell or its contents. Examples ofanalyses that can be carried out on single cells or groups of cells in apopulation can be found in U.S. Patent Publication No. 20130323732,which is incorporated herein by reference in its entirety and for thisdescription. Reagents useful in these analyses are described in U.S.Patent Publication No. 20130323732 and/or will be known to those ofskill in the art.

In some embodiments, a reaction is carried out at each capture site(separately from every other capture site), whereby the reactionproducts encode an item of capture site information. The reactionproducts can be recovered from the microfluidic device and subjected tofurther analysis. This further analysis can include the identificationof particular reaction products as having been derived from a singlecell or group of cells at a particular capture site, e.g., based, atleast in part, on the item of capture site information.

In some embodiments, the method entails incorporating nucleic acidsequences into reaction products from a cell population, wherein thenucleic acid sequences are incorporated into the reaction products ofeach cell individually or of groups of cells. In various embodiments,the nucleic acid sequences are individually incorporated into separategroups of 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 cell(s) or into groups having a number of cellswithin a range defined by any of these values. In some embodiments,these values are defined by taking an average of the number of cells percapture site. The method entails distributing cells from the populationto a plurality of capture sites in a microfluidic device so that aplurality of capture sites each comprises not more than a single cellor, where cells are to be analyzed in groups, not more than the desirednumber of cells for each group of cells. In some embodiments, thecapture sites are capable of being fluidically isolated from oneanother, for example, after cell distribution throughout the device. Incertain embodiments, the capture sites each have a capture feature thatretains the cell or group of cells in the place. In some embodiments,the capture feature resides within a chamber that can be fluidicallyisolated from other chambers within the capture site.

In some embodiments, a reaction is conducted in which nucleic acidsequences are incorporated into the reaction products of each cell orgroup of cells, individually. As those of skill in the art readilyappreciate, if the reaction is directed at intracellular templates ortargets, such as mRNA or genomic DNA, the method will typically entail acell permeabilization or lysis step to expose one or both reagents tothe intracellular template/target.

The reaction products are then recovered and analyzed in a way thatpermits the identification of particular reaction products as havingbeen derived from a single cell or group of cells at a particularcapture site. One way that this identification can be achieved is byincorporating a barcode into the reaction products. Such a barcode canencode an item of capture site information. Barcodes can be of virtuallyany length, although where the reaction products are to be subjected toDNA sequencing, shorter barcodes (e.g., 4-6 nucleotides in length) maybe preferred in some embodiments. In various embodiments, suitablebarcodes are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19,or 20 nucleotides in length or can fall within a range bounded by any ofthese values, e.g., 2-10 or 3-8.

This method finds particular application in the analysis of nucleicacids, either DNA or RNA from cells, although other molecules (proteins,carbohydrates, lipids, etc.) can be analyzed, and the method can beapplied to the analysis of any particle or group of particles (e.g.,cellular organelles, liposomes, etc.) Virtually any type of reaction orseries of reactions can be performed in the method. In certainembodiments, the reaction introduces nucleic acid sequences into thenucleic acids of a cell or group of cells. In these embodiments, thereaction may include reverse transcription, amplification, ligation orany other reaction that can be performed on a nucleic acid. Examplesinclude whole transcriptome amplification (WTA; see illustrativeembodiments shown in FIGS. 10-12), whole genome amplification (WGA; seeillustrative embodiment shown in FIG. 13), microRNA (mRNA)preamplification, target-specific amplification of RNA (see illustrativeembodiment shown in FIG. 14) or DNA (see illustrative embodiment shownin FIG. 13, wherein the template-specific portion of each primer istarget-specific), protein proximity ligation (see illustrativeembodiment shown in FIG. 16), and transposition (see illustrativeembodiment shown in FIG. 17).

The methods described herein can be used to analyze nucleic acids fromany type of cells, e.g., any self-replicating, membrane-boundedbiological entity or any non-replicating, membrane-bounded descendantthereof. Non-replicating descendants may be senescent cells, terminallydifferentiated cells, cell chimeras, serum-starved cells, infectedcells, non-replicating mutants, anucleate cells, intact nuclei, andfixed, intact (dead) cells, etc. Cells used in the methods describedherein may have any origin, genetic background, state of health, stateof fixation, membrane permeability, pretreatment, and/or populationpurity, among other characteristics. Suitable cells may be eukaryotic,prokaryotic, archaeon, etc., and may be from animals, plants, fungi,protists, bacteria, and/or the like. In illustrative embodiments, humancells are analyzed. Cells may be from any stage of organismaldevelopment, e.g., in the case of mammalian cells (e.g., human cells),embryonic, fetal, or adult cells may be analyzed. In certainembodiments, the cells are stem cells. Cells may be wildtype; natural,chemical, or viral mutants; engineered mutants (such as transgenics);and/or the like. In addition, cells may be growing, quiescent,senescent, transformed, and/or immortalized, among other states.Furthermore, cells may be a monoculture, generally derived as a clonalpopulation from a single cell or a small set of very similar cells; maybe presorted by any suitable mechanism, such as affinity binding, FACS,drug selection, etc.; and/or may be a mixed or heterogeneous populationof distinct cell types.

One advantage of the methods described herein is that they can be usedto analyze virtually any number of single cells. In various embodiments,the number of single cells analyzed can be about 10, about 50, about100, about 500, about 1000, about 2000, about 3000, about 4000, about5000, about 6000, about 7,000, about 8000, about 9,000, about 10,000,about 15,000, about 20,000, about 25,000, about 30,000, about 35,000,about 40,000, about 45,000, about 50,000, about 75,000, or about 100,000or more. In specific embodiments, the number of cells analyzed can fallwithin a range bounded by any two values listed above.

In some embodiments, this method can be carried out on a matrix-typemicrofluidic device (described further below), which facilitates theintroduction of a barcode that identifies a particular row in the deviceand a barcode that identifies a particular column, whereby thecombination uniquely identifies a particular capture site and thereforea particular cell or group of cells from which the reaction productswere derived. The method has been tested on such a device anddemonstrated to work (see results shown in FIGS. 18 and 19).

In some embodiments, each reaction can incorporate at least one UMI,which is a nucleic acid sequence that uniquely identifies the moleculeinto which it is incorporated. In variations of such embodiments, thereaction incorporates one or more barcodes in addition to one or moreUMIs. UMIs can be any length, and the length required for a givenanalysis will increase as the number of unique molecules to beidentified increases. In various embodiments, suitable UMIs are 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, or 20 nucleotides inlength or can fall within a range bounded by any of these values, e.g.,2-10, 3-8, 4-7, or 5-6.

The combined use of beads and/or sequence tags to label RNA or DNA foranalysis may avoid a need for preamplification prior to analysis andmakes the matrix-type microfluidic device reusable.

RNA Analysis

In some embodiments, the above-described methods are applied to RNAanalysis. In this case, the reaction(s) carried out at each capture sitecan include reverse transcription of RNA, e.g., second-strand synthesisto produce cDNA.

In particular methods suitable, for example, for transcriptome analysis(e.g., in single cells or in groups of cells as described above) thefirst reagent(s) can include a reverse transcription (RT) primer. Anillustrative RT primer is shown in FIG. 10 (“3′ RT primer”). This RTprimer includes a first barcode (“bc”) 5′ of a poly-dT sequence. Thepoly-dT sequence should be sufficiently long to anneal to the poly-Atails of mRNA, typically on the order of 18-30 nucleotides in length.This RT primer optionally includes a first UMI, which is also preferably5′ of the poly-dT sequence. FIG. 10 shows the first barcode 5′ of theUMI; however, those of skill in the art appreciate that the order ofthese elements may be reversed. As shown in FIG. 10, the RT primer canadditionally include a first linker, preferably at the 5′ end of the RTprimer. The first linker can be used to facilitate unbiasedamplification and, wherein DNA sequencing is to be carried out, 3′ endenrichment after tagmentation. In some embodiments, the RT primeradditionally comprises an anchor sequence 3′ of the poly-dT sequence.The length and composition of the anchor sequence can vary, and theselection of a suitable anchor sequence for a particular analysis iswithin the level of skill in the art. Typically, the anchor sequence isat least two nucleotides in length.

In some embodiments, the second reagent(s) comprise a 5′ oligonucleotidecomprising a poly-riboG sequence. An illustrative oligonucleotide ofthis type is shown in FIG. 10. This oligonucleotide is identified as“TSO oligo” for “template-switching oligonucleotide.” This 5′oligonucleotide can include a second barcode 5′ of the poly-riboGsequence (shown, e.g., in FIG. 10 as one of the two bars between thepoly-riboG sequence and a linker). The 5′ oligonucleotide can optionallyinclude a second UMI, which can be the same as or different from thefirst UMI, which is also preferably 5′ of the poly-riboG sequence(shown, e.g., in FIG. 10 as the other to the two bars between thepoly-riboG sequence and the linker). As shown in FIG. 10, the 5′oligonucleotide can additionally include a second linker, preferably atthe 5′ end of the 5′ oligonucleotide. The second linker can be the sameas or different from the first linker, and like the first linker, can beused to facilitate unbiased amplification.

In particular embodiments, the method is carried out in a microfluidicdevice, as described below. In this case, the RT primer can be deliveredto capture sites via one set of the input lines, and the 5′oligonucleotide can be delivered to the capture sites via the other setof input lines.

In certain embodiments, the use of these two reagents in one of theabove-described methods produces cDNA wherein one strand has thestructure: 5′-second linker-nucleotide sequence derived from RNA-firstlinker-3′, with at least one barcode located in between the linkers. Ina variation of this embodiment, the first barcode is located adjacent tothe first linker and/or the second barcode is located adjacent to thesecond linker. For example, one strand of the cDNA can have thestructure:

-   -   3′-second linker-poly dC-nucleotide sequence derived from        RNA-first barcode-first linker-5′.

Where a second barcode is included, one strand of the cDNA can have thestructure:

-   -   3′-second linker-second barcode-poly dC-nucleotide sequence        derived from RNA-first barcode-first linker-5′.

The inclusion of a UMI can produce, for example:

-   -   3′-second linker-poly dC-nucleotide sequence derived from        RNA-first UMI-first barcode-first linker-5′; or    -   3′-second linker-poly dC-nucleotide sequence derived from        RNA-first barcode-first UMI-first linker-5′.

And the inclusion of second UMI can produce, for example:

-   -   3′-second linker-second barcode-second UMI-poly dC-nucleotide        sequence derived from RNA-first UMI-first barcode-first        linker-5′;    -   3′-second linker-second barcode-second UMI-poly dC-nucleotide        sequence derived from RNA-first barcode-first UMI-first        linker-5′;    -   3′-second linker-second UMI-second barcode-poly dC-nucleotide        sequence derived from RNA-first UMI-first barcode-first        linker-5′; or    -   3′-second linker-second UMI-second barcode-poly dC-nucleotide        sequence derived from RNA-first barcode-first UMI-first        linker-5′.

DNA Analysis

As those of skill in the art appreciate, the chemistry described abovefor RNA analysis can be adapted to DNA analysis, e.g., where thereaction(s) carried out at each capture site includes amplification ofDNA.

In particular DNA amplification embodiments, the first and/or secondreagent(s) include first and/or second amplification primers,respectively, wherein the first and/or second amplification primerscomprise a first or second barcode, respectively, that is 5′ of a primersequence. The primer sequence(s) can be random or designed to amplify aparticular target nucleic acid (i.e., “target-specific”). In someembodiments, the first and/or second amplification primers mayadditionally include a first or second UMI, respectively. Any UMI ispreferably 5′ of the primer sequence. In some embodiments, the firstand/or second amplification primer additionally includes a first orsecond linker, preferably at the 5′ end(s) of the primer(s), e.g., tofacilitate unbiased amplification. The discussion above regardingsuitable lengths and sequences for barcodes and UMIs apply equally inthe DNA analysis context. For primers including barcodes and UMIs, theirpositions relative to one another are not critical.

In particular embodiments, DNA amplification is carried out in amicrofluidic device, as described below. In this case, the firstamplification primer can be delivered to capture sites via one set ofthe input lines, and the second amplification primer can be delivered tothe capture sites via the other set of input lines.

In certain embodiments, the use of two such amplification primers in oneof the above-described methods produces an amplicon, wherein one strandhas the structure: 5′-second linker-nucleotide sequence derived fromsample DNA-first linker-3′, with a barcode located in between thelinkers. For example, one strand of the amplicon can have the structure:

-   -   3′-second linker-nucleotide sequence derived from sample        DNA-first barcode-first linker-5′.

Where a second barcode is included, one strand of the amplicon can havethe structure:

-   -   3′-second linker-second barcode-nucleotide sequence derived from        sample DNA-first barcode-first linker-5′.

The inclusion of a UMI can produce, for example:

-   -   3′-second linker-nucleotide sequence derived from sample        DNA-first UMI-first barcode-first linker-5′; or    -   3′-second linker-nucleotide sequence derived from sample        DNA-first barcode-first UMI-first linker-5′.

And the inclusion of second UMI can produce, for example:

-   -   3′-second linker-second barcode-second UMI-nucleotide sequence        derived from sample DNA-first UMI-first barcode-first linker-5′;    -   3′-second linker-second barcode-second UMI-nucleotide sequence        derived from sample DNA-first barcode-first UMI-first linker-5′;    -   3′-second linker-second UMI-second barcode-nucleotide sequence        derived from sample DNA-first UMI-first barcode-first linker-5′;        or    -   3′-second linker-second UMI-second barcode-poly dC-nucleotide        sequence derived from sample DNA-first barcode-first UMI-first        linker-5′.

In any of the above-described methods, all of the method steps can, butneed not, be performed in a microfluidic device.

Any of these methods can optionally include preamplification, mostconveniently, after addition of linkers to either end of the cDNA orDNA. For example, preamplification can be carried out to increase thelevels of the cDNA or amplicons before further characterization (suchas, e.g., DNA sequencing). Preamplication can be carried out usinglinker primers that anneal to the first and second linkers, wherein thelinker primers are the same or different (depending on whether thelinkers themselves are the same or different). Preamplification can becarried out in the microfluidic device or after exporting reactionproducts from the device.

In some embodiments, any of the above methods can be carried out toprepare templates for DNA sequencing. In such embodiments, the reactionperformed in the microfluidic device can incorporate one or more DNAsequencing primer binding sites into the reaction products, or thesesites can be incorporated into the reaction products after export fromthe microfluidic device. In specific embodiments, DNA sequencing primerbinding sites can be added by tagmentation, which is a well-knowntransposase-based in vitro shotgun method in which the DNA to besequenced is simultaneously fragmented and tagged with transposon endsto introduce sequences that facilitate subsequent sequencing.

Accordingly, in some embodiments, the methods include subjecting thereaction products to DNA sequencing, e.g., Sanger sequencing,next-generation sequencing (e.g., bridge sequencing), orthird-generation sequencing. In variations of such embodiments, thesequences obtained from DNA sequencing can be identified as having beenderived from a particular capture site based on one or two barcodes.

As discussed in more detail below, reaction products from a particularrow or column of a matrix-type microfluidic device can be exported as apool. Any subsequent characterization of reaction products, such as DNAsequencing, can be carried out on individual exported pools. However, itis also contemplated that the pools themselves can be pooled prior tofurther characterization. In this case, the reaction product(s) fromeach separate capture site in the microfluidic device is typicallydistinct, which is readily achieved, e.g., by using two barcodesequences to encode the row and column location of the capture site inthe microfluidic device.

Primer Combinations

Any of the primers or oligonucleotides described above may be combinedto form primer combinations. Typically, primer combinations include 2,3, 4, or more primers or oligonucleotides that are used together in amethod such as those described herein.

For example, a primer combination for use in producing cDNA from RNA(first strand synthesis) can include:

(a) a reverse transcription (RT) primer including an anchor sequence, apoly-dT sequence 5′ of the anchor sequence, a first barcode 5′ of thepoly-dT sequence, and a first linker 5′ of the first barcode sequence;and

(b) a 5′ oligonucleotide including a poly-riboG sequence, a secondbarcode 5′ of the poly-riboG sequence, and a second linker 5′ of thesecond barcode.

In certain embodiments, one or both of these primers can include a UMI.

An illustrative primer combination for use in amplifying DNA can includefirst and second amplification primers that each include: a primersequence; a barcode that is 5′ of the primer sequence, wherein thebarcodes in each primer are different; and a linker that is 5′ of thebarcode; wherein one or both primers also include a UMI that is 5′ ofthe primer sequence and 3′ of the linker.

These primer combinations can also include one or more linker primer(s)that anneal(s) to the linkers, e.g., to facilitate unbiasedamplification. In some embodiments, the combination includes two linkerprimers: a 5′ linker primer and a different 3′ linker primer. In someembodiments requiring preamplification of cDNA or amplicons producedusing the above primer combinations, one or both linker primers can beused to carry out this preamplification.

A primer combination intended for use in preparing DNA sequencingtemplates for bridge sequencing can optionally include a primerincluding a portion specific for the 3′ linker primer or its complementand/or a primer including a portion specific for the 5′ linker primer orits complement, wherein the primer(s) additionally include a flow cellsequence useful in cluster generation in bridge sequencing. The flowcell sequence is generally 5′ of the linker-specific portion.

Matrix-Type Microfluidic Devices

In certain embodiments, a matrix-type microfluidic device useful in themethod described above includes capture sites arranged in a matrix of Rrows and C columns, wherein R and C are integers greater than 1. Eachcapture site can include a capture feature that is capable of capturingjust one cell or, where cells are to be analyzed in groups, not morethan the desired number of cells for each group of cells. The capturesites can be fluidically isolated from one another after distribution ofcells to the capture sites. The device also includes a set of R firstinput lines configured to deliver the first reagent(s) to capture sitesin a particular row, and a set of C second input lines configured todeliver second reagent(s) to capture sites in a particular column,wherein this delivery is separate from the delivery first reagent(s). Anillustrative device of this type is shown schematically in FIG. 1A. FIG.1B illustrates the delivery of R different barcodes through the Rdifferent first input lines to the capture sites. FIG. 1C illustratesthe delivery of C different barcodes through C different input lines tothe capture sites. In particular embodiments, all barcodes will beunique, i.e., different from every other barcode provided to the device.FIG. 1D illustrates that, after the reaction has been carried out,reaction products can be harvested from each column as a pool, forexample, by applying a harvesting fluid to the C second input lines topush the reaction products out of outlets at one end of the input lines.(Those of skill in the art readily appreciate that reaction productscould alternatively be harvested by row in the same manner in adifferent implementation.) FIG. 2 shows a photograph of the device shownschematically in FIG. 1.

In certain embodiments, the matrix-type microfluidic device permitsanalysis of individual cells or groups of cells, e.g., up to (andincluding) 1000. The cells can be intact or partially or fully disrupted(e.g., permeablized or lysed) after capture or isolation of one or morecells at each capture site. In the latter case, the device is configuredto provide this functionality (see, e.g., FIG. 11). In some embodiments,the device is transparent on at least one surface to permit imaging tovisualize cell number or phenotype (where the cells or their contentshave been reacted with an optically detectable label). In someembodiments, the device is configured to perform “X-Y” combinatorialbarcoding, whereby reaction products may be exported in one or morepools (which may themselves be pooled) and further analyzed in multiplex(e.g., by amplification), followed by “de-multiplexing” (“demux”) toassign particular reaction products to particular capture sites. Thistype of barcoding is illustrated in FIG. 1, which shows the same set of3′ barcodes (“3′BC” in FIG. 1B) being delivered to each column and thesame set of 5′ barcodes (“5′BC” in FIG. 1C) being delivered to each row.

FIG. 3 shows a photomicrograph of an illustrative capture featuresuitable for capturing a single cell in such a device, as well as anillustrative flowchart for a method in which barcodes are added by rowand column into the reaction products from the captured cell. FIG. 4Ashows a series of increasingly more miniaturized capture features, whichfacilitate the analysis of more cells per chip. FIG. 4B shows thearrangement of capture features in rows and columns, together with thechannels for distributing cells to the capture features. FIG. 4C showsphotomicrographs of the miniaturized capture features.

The table in FIG. 4D reports the cell occupancy data for two differentcapture site designs, with the top panel showing the results for theminiaturized features, and the bottom panel showing the results fordouble grooves capture features with different height ratios for top andbottom grooves. The first column in the table in FIG. 4D reports thenumber of capture features having no cells, and second column reportsthe number of capture features having just 1 cell. FIG. 5 shows aschematic of the double grooves capture features including the channelsfor distributing cells to the capture features. This geometry allows formore capture sites per unit area. FIG. 6A shows the flow resistance forthe main and bypass flow in such a device, illustrated schematically inFIG. 6B and in photomicrographs in FIGS. 6C and D. FIG. 7 illustratesthe activation of bypass peristaltic pumping in such a device,schematically in panel A and in a photomicrograph in panel B. FIGS. 8Aand B illustrates closing the main flow output in such a device,schematically in panel A and in a photomicrograph in panel B. The tablein FIG. 8C reports cell occupancy data for the double grooves capturefeature design, with number of capture features having no cells incolumn 1 of the table, and the number of capture features having just 1cell in column 2 of the table.

FIG. 9 shows an illustrative unit capture site with four chambers, eachof which is available for reagent(s). The capture feature is located inone of these four chambers. In use, a first reagent(s) can be loadedinto the capture site from bottom to top (purple arrow), and a secondreagent can be loaded into a capture site from left to right (greenarrow).

In various embodiments, a microfluidic device having from about 97 toabout 1000 separate capture sites is employed to carry out one or moreof the methods described herein, particularly from about 97 to about9000 capture sites, more particularly from about 97 to about 8000capture sites, and even more particularly from about 97 to about 7500capture sites. In some embodiments the microfluidic device can havegreater than 100, greater than 200, greater than 300, greater than 400,greater than 500, greater than 600, greater than 700, greater than 800,greater than 900, or greater than 1000 capture sites.

In some embodiments, the capture sites have one or more reactionchambers ranging from about 2 nL to about 500 nL. The lower the reactionchamber volume, the higher the effective concentration of any targetnucleic acid. In certain embodiments, the reaction chamber is from about2 nL to about 50 nL, preferably 2 nL to about 25 nL, more preferablyfrom about 4 nL to about 15 nL. In some embodiments, the reactionchamber volume is 5 nL, 6, nL, 7 nL, 8 nL, 9 nL, 10 nL, 11 nL, or 12 nL,or falls within any range bounded by any of these values.

Microfluidic devices meeting the specifications described herein, andsystems employing them the carry out the disclosed method can bedesigned and fabricated based on the guidance herein and in priorco-owned patent publications, such as U.S. Patent Publication No.2013/0323732, published May 12, 2013, Anderson et al. (herebyincorporated by reference for their descriptions of single-cell analysismethods and systems). For example, the C₁™ Single-Cell Auto Prep Systemavailable from Fluidigm Corporation (South San Francisco, Calif.)provides bench-top automation of the multiplexed isolation, lysis, andreactions on nucleic acids from single cells in an IFC™. In particular,the C₁ Single-Cell Auto Prep Array™ IFC is a matrix-type microfluidicdevice that facilitates capture and highly paralleled preparation of 96individual cells. When used properly, each capture site within the chipcaptures one single cell. Sometimes, a site may capture zero, two, ormore cells; however, the exact number of captured cells in each capturedsite of a C₁ chip is easily verified at high confidence and easilydocumented in a microscopic picture. In certain embodiments, cells arecaptured and barcoding is carried out in each separate reaction volumeto produce barcoded nucleic acid molecules, which are analyzed, mostconveniently by DNA sequencing, be it Sanger sequencing, next-generationsequencing, or third-generation sequencing, optionally afterpreamplification.

Kits

Kits according to the invention can include one or more reagents usefulfor practicing one or more methods described herein. A kit generallyincludes a package with one or more containers holding the reagent(s),as one or more separate compositions or, optionally, as admixture wherethe compatibility of the reagents will allow. The kit can also includeother material(s) that may be desirable from a user standpoint, such asa buffer(s), a diluent(s), a standard(s), and/or any other materialuseful in sample processing, washing, or conducting any other step ofthe assay. In specific embodiments, the kit includes one or morematrix-type microfluidic devices and/or primers/oligonucleotidesdiscussed above or combinations thereof.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

In addition, all other publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

EXAMPLES Example 1 High-Throughput (HT) IFC™

Various designs for a high-throughput (HT) IFC™ are shown in theaccompanying figures. One aspect of the HT IFC™ is that it containsmodified (miniaturized) capture features that enable eight times thenumber of capture sites in the same area as a normal IFC™.

Another aspect of the HT IFC™ is that it enables the multiplexing ofbarcodes and this, combined with companion chemistry, such as thatdescribed below, allows the HT IFC™ to go beyond the current 96single-cell limit of C1™ system. Specifically, the HT IFC™ canindividually address each chamber with up to two inputs, permitting theseparate addition of at least two different barcodes in discrete liquidadditions to a single cell.

Example 2 Companion Chemistry for HT IFC™ for Transcriptome Analysis

The chemistry to enable barcoding and cell de-multiplexing on an HT IFC™can include a set of modified oligonucleotides that allow single-celltranscriptome identification (for messenger [poly-A] RNA) and 3′ endcounting of the transcripts used with conventional,commercially-available reverse transcriptase enzymes (MuMLV Rnase Hactivity mutants) and conventional TaqPolymerases, as well as theNextera XT™ kit. The set can include one or more of the following (seeFIG. 10, wherein the numbers correspond to the oligonucleotide numbersgive below):

1. An oligonucleotide (RT primer, referred to as a ‘row’ barcode in thecontext of the HT IFC™) directed at the 3′ end of an mRNA transcriptminimally including an 2-nucleotide anchor sequence, a poly-dT sequence(18-30 dTs), a chamber identification barcode of between 4-6nucleotides, an optional randomer of 5-6 nucleotides for single moleculeidentification (UMI), and a linker sequence for unbiased amplificationand 3′ end enrichment after tagmentation.

2. An oligonucleotide that allows completion of the cDNA molecule fromthe 5′ end of the transcript and linker add-on for unbiasedamplification. This oligonucleotide includes an optional 4-5-nucleotidebarcode as well as an optional 5-nucleotide randomer for single moleculeidentification (UMI) along with the linker sequence for amplification atthe 5′ end.

3. An oligonucleotide that facilitates preamplification of the linkersappended to either end of the first strand of cDNA.

4. An oligonucleotide (cluster 2) specific for the 3′ end linker thatallows for enrichment of the 3′ end of the transcript followingtagmentation, used during the addition of the flow cell sequence. Anoptional oligonucleotide directed at the 5′ linker to enrich for the 5′end of the transcript may also be used.

The HT IFC™ and the oligonucleotides, used together, allow the export ofa plentitude of cDNA material barcoded (row by row) for individual cells(barcoded by exported pools) that can then be prepared in pools for useon a second generation sequencing platform or otherwise analyzed In theversion illustrated herein, the HT IFC™ plus companion chemistryincreases the number of cells that can be queried on a single chip morethan 8-fold compared to the best currently available throughput, whileat the same time significantly reducing the number of librarypreparation reactions from a potential 800 single reactions off-chip toonly 20.

1. A method of exposing cells from a population to at least twodifferent reagents, wherein each cell is exposed to the reagentsindividually, or in groups of two of more, the method comprising: (a)distributing cells from the population to a plurality of capture sitesin a microfluidic device so that a plurality of capture sites eachcomprises one or more cells; (b) providing one or more first reagent(s)to each capture site; (c) providing one or more second reagent(s) toeach capture site, wherein the second reagent(s) is/are different fromthe first reagent(s) and is/are provided separately from the firstreagent(s); (d) conducting a reaction, whereby the reaction productsencode an item of capture site information; (e) recovering the reactionproducts; and (f) analyzing the reaction products, wherein such analysispermits the identification of particular reaction products as havingbeen derived from a single cell or group of cells at a particularcapture site.
 2. A method of incorporating nucleic acid sequences intoreaction products from a cell population, wherein the nucleic acidsequences are incorporated into the reaction products of each cellindividually, or in groups of up to 1000, the method comprising: (a)distributing cells from the population to a plurality of capture sitesin a microfluidic device so that a plurality of capture sites eachcomprises one or more cells; (b) providing one or more first reagent(s)to each capture site; (c) providing one or more second reagent(s) toeach capture site, wherein the second reagent(s) is/are different fromthe first reagent(s) and is/are provided separately from the firstreagent(s); (d) conducting a reaction in which nucleic acid sequencesare incorporated into the reaction products of each cell or group ofcells, individually; (e) recovering the reaction products; and (f)analyzing the reaction products, wherein such analysis permits theidentification of particular reaction products as having been derivedfrom a single cell or group of cells at a particular capture site.
 3. Amethod of incorporating nucleic acid sequences into nucleic acids of acell population, wherein the nucleic acid sequences are incorporatedinto the nucleic acids of each cell individually or in groups of up to1000, the method comprising: (a) distributing cells from the populationto a plurality of capture sites in a microfluidic device so that aplurality of capture sites each comprises one or more cells; (b)providing one or more first reagent(s) to each capture site; (c)providing one or more second reagent(s) to each capture site, whereinthe second reagent(s) is/are different from the first reagent(s) andis/are provided separately from the first reagent(s); (d) conducting areaction in which nucleic acid sequences are incorporated into thenucleic acids of each cell or group of cells, individually, to producereaction products; (e) recovering the reaction products; and (f)analyzing the reaction products, wherein such analysis permits theidentification of particular reaction products as having been derivedfrom a single cell or group of cells at a particular capture site. 4.The method of claim 1, where the distribution is carried out so that aplurality of capture sites each comprise not more than a single cell. 5.The method of claim 1, wherein the reaction incorporates a nucleotidebarcode into the reaction products.
 6. The method of claim 5, whereinthe barcode encodes an item of capture site information.
 7. The methodof claim 1, wherein the reaction incorporates a nucleic acid sequencethat uniquely identifies the molecule into which it is incorporated(UMI).
 8. The method of claim 3, wherein the reaction comprises reversetranscription of RNA.
 9. The method of claim 8, wherein the firstreagent(s) comprise a reverse transcription (RT) primer comprising apoly-dT sequence and a first barcode 5′ of the poly-dT sequence.
 10. Themethod of claim 9, wherein the RT primer additionally comprises a firstUMI.
 11. The method of claim 10, wherein the first UMI is 5′ of thepoly-dT sequence.
 12. The method of claim 9, wherein the RT primeradditionally comprises a first linker.
 13. The method of claim 12,wherein the first linker is at the 5′ end of the RT primer.
 14. Themethod of claim 9, wherein the RT primer additionally comprises ananchor sequence 3′ of the poly-dT sequence.
 15. The method of claim 8,wherein the reaction additionally comprises second-strand synthesis toproduce cDNA.
 16. The method of claim 8, wherein the second reagent(s)comprise a 5′ oligonucleotide comprising a poly-riboG sequence.
 17. Themethod of claim 15, wherein the 5′ oligonucleotide comprises a secondbarcode 5′ of the poly-riboG sequence.
 18. The method of claim 15,wherein the 5′ oligonucleotide additionally comprises a second UMI. 19.The method of claim 18, wherein the second UMI is 5′ of the poly-riboGsequence.
 20. The method of claim 15, wherein the 5′ oligonucleotideadditionally comprises a second linker.
 21. The method of claim 20,wherein the second linker is at the 5′ end of the 5′ oligonucleotide.22. The method of claim 21, wherein the method comprises producing cDNA,wherein one strand has the structure: 5′-second linker-nucleotidesequence derived from RNA-first linker-3′, with a barcode located inbetween the linkers.
 23. The method of claim 22, wherein the firstbarcode is located adjacent to the first linker.
 24. The method of claim23, wherein the second barcode is located adjacent to the second linker.25. The method of claim 23, wherein said one strand of cDNA has thestructure: 3′-second linker-poly dC-nucleotide sequence derived fromRNA-first barcode-first linker-5′.
 26. The method of claim 25, whereinsaid one strand of cDNA has the structure: 3′-second linker-secondbarcode-poly dC-nucleotide sequence derived from RNA-first barcode-firstlinker-5′.
 27. The method of claim 25, wherein said one strand of cDNAhas a structure selected from the group consisting of: 3′-secondlinker-poly dC-nucleotide sequence derived from RNA-first UMI-firstbarcode-first linker-5′; and 3′-second linker-poly dC-nucleotidesequence derived from RNA-first barcode-first UMI-first linker-5′. 28.The method of claim 27, wherein said one strand of cDNA has a structureselected from the group consisting of: 3′-second linker-secondbarcode-second UMI-poly dC-nucleotide sequence derived from RNA-firstUMI-first barcode-first linker-5′; 3′-second linker-secondbarcode-second UMI-poly dC-nucleotide sequence derived from RNA-firstbarcode-first UMI-first linker-5′; 3′-second linker-second UMI-secondbarcode-poly dC-nucleotide sequence derived from RNA-first UMI-firstbarcode-first linker-5′; and 3′-second linker-second UMI-secondbarcode-poly dC-nucleotide sequence derived from RNA-first barcode-firstUMI-first linker-5′.
 29. The method of claim 3, wherein the reactioncomprises amplification of DNA.
 30. The method of claim 29, wherein thefirst and/or second reagent(s) comprise first and/or secondamplification primers, respectively, wherein the first and/or secondamplification primers comprise a first or second barcode, respectively,that is 5′ of a primer sequence.
 31. The method of claim 30, wherein thefirst and/or second amplification primers additionally comprise a firstor second UMI, respectively.
 32. The method of claim 31, wherein thefirst or second UMI is 5′ of the primer sequence.
 33. The method claim30, wherein the first and/or second amplification primer additionallycomprises a first or second linker.
 34. The method of claim 33, whereinthe first or second linker is at the 5′ end of the amplification primer.35. The method of claim 34, wherein the method comprises producingamplicons, wherein one strand has the structure: 5′-secondlinker-nucleotide sequence derived from cellular DNA-first linker-3′,with a barcode located in between the linkers.
 36. The method of claim35, wherein one strand has the structure: 3′-second linker-nucleotidesequence derived from cellular DNA-first barcode-first linker-5′. 37.The method of claim 25, wherein said one strand has the structure:3′-second linker-second barcode-nucleotide sequence derived fromcellular DNA-first barcode-first linker-5′.
 38. The method of claim 25,wherein said one strand has a structure selected from the groupconsisting of: 3′-second linker-nucleotide sequence derived fromcellular DNA-first UMI-first barcode-first linker-5′; and 3′-secondlinker-nucleotide sequence derived from cellular DNA-first barcode-firstUMI-first linker-5′.
 39. The method of claim 27, wherein said one strandhas a structure selected from the group consisting of: 3′-secondlinker-second barcode-second UMI-nucleotide sequence derived fromcellular DNA-first UMI-first barcode-first linker-5′; 3′-secondlinker-second barcode-second UMI-nucleotide sequence derived fromcellular DNA-first barcode-first UMI-first linker-5′; 3′-secondlinker-second UMI-second barcode-nucleotide sequence derived fromcellular DNA-first UMI-first barcode-first linker-5′; and 3′-secondlinker-second UMI-second barcode-poly dC-nucleotide sequence derivedfrom cellular DNA-first barcode-first UMI-first linker-5′.
 40. Themethod of claim 1, wherein the microfluidic device comprises amatrix-type microfluidic device comprising: capture sites arranged in amatrix of R rows and C columns, wherein R and C are integers greaterthan 1, and wherein the capture sites can be fluidically isolated fromone another after distribution of cells to the capture sites; a set of Rfirst input lines configured to deliver the first reagent(s) to capturesites in a particular row; a set of C second input lines configured todeliver second reagent(s) to capture sites in a particular column,wherein said delivery is separate from the delivery first reagent(s),wherein, after the reaction, reaction products are recovered from themicrofluidic device in pools of reaction products from individual rowsor columns.
 41. The method of claim 40, wherein an RT primer isdelivered to capture sites via one set of the input lines, and a 5′oligonucleotide is delivered to the capture sites via the other set ofinput lines.
 42. The method of claim 40, wherein a first amplificationprimer is delivered to capture sites via one set of the input lines, anda second amplification primer is delivered to the capture sites via theother set of input lines.
 43. The method of claim 1, wherein all methodssteps are performed in the microfluidic device.
 44. The method of claim1, wherein the reaction products are subjected to preamplification usinglinker primers that anneal to the first and second linkers, wherein thelinker primers are the same or different.
 45. The method of claim 44,wherein said preamplification is performed in the microfluidic device.46. The method of claim 1, wherein the reaction products are subjectedto tagmentation.
 47. The method of claim 1, wherein the reactionincorporates one or more DNA sequencing primer binding sites into thereaction products.
 48. The method of claim 1, wherein the reactionproducts are subjected to DNA sequencing.
 49. The method of claim 48,wherein the sequences obtained from DNA sequencing are identified ashaving been derived from a particular capture site based on one or twobarcodes.
 50. The method of claim 40, wherein the exported pools areseparately subjected to preamplification using linker primers thatanneal to the first and second linkers, wherein the linker primers arethe same or different.
 51. The method of claim 40, wherein the exportedpools are combined into one reaction mixture, which is subjected topreamplification using linker primers that anneal to the first andsecond linkers, wherein the linker primers are the same or different.52. The method of claim 40, wherein the microfluidic device issufficiently transparent on at least one surface to permit visualizationof cells and/or, when a visualizable label is employed, signalsassociated with cells or reaction products.
 53. The method of claim 52,additionally comprising imaging the cell-occupied capture sites beforeconducting the reaction.
 54. The method of claim 1, wherein the reactioncomprises whole transcriptome amplification (WTA), whole genomeamplification (WGA), protein proximity ligation, microRNA (mRNA)preamplification, target-specific amplification of RNA or DNA.
 55. Themethod of claim 1, wherein the microfluidic device comprises at least750 capture sites.
 56. A matrix-type microfluidic device comprising: aplurality of capture sites arranged in a matrix of R rows and C columns,wherein R and C are integers greater than 1, and wherein: each capturesite comprises a capture feature that captures one or more cells; thecapture sites can be fluidically isolated from one another afterdistribution of cells to the capture sites; and a set of R first inputlines configured to deliver the first reagent(s) to capture sites in aparticular row; and a set of C second input lines configured to deliversecond reagent(s) to capture sites in a particular column, wherein saiddelivery is separate from the delivery first reagent(s).
 57. The deviceof claim 56, wherein the capture feature is configured to capture notmore than a single cell.
 58. The device of claim 56, wherein themicrofluidic device is sufficiently transparent on at least one surfaceto permit visualization of cells and/or, when a visualizable label isemployed, signals associated with cells or reaction products.
 59. Thedevice of claim 56, wherein each capture site comprises four chambersthat can be fluidically isolated from one another, wherein one of saidchambers comprises the capture feature.
 60. A method of operating themicrofluidic device of claim 56, wherein the method comprises: (a)distributing cells from a population of cells to the capture sites sothat a plurality of capture sites comprise one or more cells; (b) afterdistribution, fluidically isolating the capture sites from one another;(c) providing one or more first reagent(s) to each fluidically isolatedcapture site via the R first input lines; (d) providing one or moresecond reagent(s) to each fluidically isolated capture site via the Csecond input lines, wherein the second reagent(s) is/are different fromthe first reagent(s); and (e) conducting a reaction.
 61. The method ofclaim 60, wherein a plurality of capture sites comprise not more than asingle cell.
 62. The method of claim 60, additionally comprisingrecovering the reaction products as a pool of reaction products fromeach row or as a pool of reaction products from each column.
 63. Themethod of claim 60, wherein said recovering comprises providing aharvesting reagent to the R first input lines or the C second inputlines.
 64. A primer combination for use in producing cDNA from RNA, thecombination comprising: (a) a reverse transcription (RT) primercomprising an anchor sequence, a poly-dT sequence 5′ of the anchorsequence, a first barcode 5′ of the poly-dT sequence, and a first linker5′ of the first barcode sequence; and (b) a 5′ oligonucleotidecomprising a poly-riboG sequence, a second barcode 5′ of the poly-riboGsequence, and a second linker 5′ of the second barcode.
 65. The primercombination of claim 64, wherein one or both primers comprise a UMI. 66.A primer combination for use in amplifying DNA, the combinationcomprising first and second amplification primers that can prime theproduction of an amplicon in the presence of suitable template DNA,wherein each amplification primer comprises: a primer sequence; abarcode that is 5′ of the primer sequence, wherein the barcodes in eachprimer are different; and a linker that is 5′ of the barcode; whereinone or both primers also comprise a UMI that is 5′ of the primersequence and 3′ of the linker.
 67. The primer combination of claim 64,additionally comprising one or more linker primer(s) that anneal(s) tothe linkers.
 68. The primer combination of claim 64, wherein the linkerprimers comprise a 5′ linker primer and a different 3′ linker primer.69. The primer combination of claim 68, additionally comprising a primercomprising a portion specific for the 3′ linker primer or its complementand/or a primer comprising a portion specific for the 5′ linker primeror its complement, wherein the primer(s) additionally comprise a flowcell sequence useful in cluster generation in bridge sequencing.
 70. Amethod of producing cDNA from RNA, wherein the primer combination ofclaim 64 is employed for first-strand synthesis.
 71. A method ofamplifying DNA, the method comprising contacting template DNA with theprimer combination of claim 66 to produce amplicons.
 72. A method ofpreamplifying the cDNA or amplicons of claim 70, respectively, themethod comprising preamplifying the cDNA or amplicons with one or morelinker primer(s) that anneal(s) to the linkers.
 73. A method of clustergeneration in bridge sequencing of cDNA or amplicons produced in claim70, the method comprising conducting cluster generation using a primercomprising a portion specific for a 3′ linker primer or its complementand/or a primer comprising a portion specific for a 5′ linker primer orits complement, wherein the primer(s) additionally comprise a flow cellsequence useful in cluster generation in bridge sequencing.